There are certain situations where it is desirable to vascularize (to form blood vessels in) the tissue of a living animal.
One such situation is in the treatment of heart muscle after an infarction, or heart attack. Depending on the severity of the infarction, some of the heart muscle will be damaged ("ischemic") because of the inability of the remaining coronary blood supply to provide enough oxygen and nutrients to it.
Considerable effort has been directed to the use of therapeutic agents such as basic fibroblast growth factor (bFGF) or V-endothelial growth factor (VEGF) (S. Takeshita et al, J. Clin Invest. 1994, 93: 662-670) to generate a collateral blood supply to damaged heart tissue after a period of ischemia and thereby aid in the recovery of the heart after a heart attack. These therapeutic agents are expensive, somewhat unstable and difficult to deliver and have some undesirable side effects. The disadvantage exists that the generation of new blood vessels in ischemic heart muscle is not presently possible without the use of drugs.
Another such situation in which vascularization is desirable is the treatment of peripheral ischemia that may result, for example, as a complication of Diabetes. The microcirculation supplying the peripheral tissue is not able to provide adequate oxygen and nutrients to the legs and feet which results in tissue death, susceptibility to infection, and eventually, amputation.
A further situation is in the treatment of various wounds, including severe burns, or chronic wounds such as bed sores or venous and diabetic ulcers. Chronic wounds are difficult to heal, partly due to an insufficient vascular bed supplying nutrients and healing factors to the wound site.
Another situation is the treatment of the healing interfaces between transplanted and host tissue. For example, the incorporation and survival of cadaverous gum tissue, implanted to replace diseased gum tissue, is hindered by a lack of blood vessels supplying the new tissue with nutrients.
Another situation in which it is desirable to generate new blood vessels is in the field of implantable drug delivery systems, such as microencapsulated animal cells which produce and release a therapeutic agent, such as a biologically active molecule, to the host in which they are implanted.
When any material or device is implanted in the body of an animal (as used herein, terms "animal" and "host" include humans), the body responds by producing what is termed a foreign body reaction. This involves various leukocytes, particularly macrophages and neutrophils, and typically results in an avascular fibrous capsule that is intended to isolate or "wall off" the material or device from the body.
This reaction is appropriate for many situations but not for implantable drug delivery systems, such as implantable microcapsules, where it is preferable to have blood vessels present close to the surface of the material comprising the delivery system. These blood vessels are then able to carry the therapeutic agent to parts of the body where it is needed. An avascular fibrous capsule formed around the drug delivery system acts as a diffusion barrier between the drug delivery system and the body's blood vessels, preventing or at least slowing the delivery of the therapeutic agent.
The development of fibrogenic tissue around microcapsules has been a persistent and fatal problem with prior art drug delivery systems utilizing microcapsules. In order to survive, implanted cells require free diffusion of nutrients, gases, and waste products while releasing to the host the intended cellular product produced by the microencapsulated cells. However, the formation of scar tissue and lack of vascularization leave the implanted cells effectively cut off from the nutrients necessary for their survival. Therefore, a disadvantage exists that no drug delivery systems utilizing implanted microcapsules are known to date which are vascularized (i.e., create blood vessels in the surrounding tissue in the immediate vicinity of the capsules) when implanted into a host animal.
In order to further promote effective diffusion of essential nutrients, toxic metabolic end-products and cellular products through the microcapsule wall, it is preferred that cells be encapsulated in small microcapsules (defined as microcapsules less than 500 .mu.m in diameter) because of their high surface area and thin walls.
Various techniques have been used for encapsulating mammalian cells in small microcapsules. For example, alginate-polylysine microcapsules having diameters between 250 .mu.m and 350 .mu.m have been prepared with an electrostatic droplet generator, as reported by Sun et al., ASAIO J. 38:125-127 (1992) and Halle et al., Transplant. Proc. 24:2930-2932 (1992). Alginate-polylysine microcapsules having a diameter of less than 500 .mu.m have been produced with an air-jet droplet generator as reported by Wolters et al., J. Appl. Biomat. 3:281-286 (1992).
Zekorn et al., Acta Diabetol. 29:41-45 (1992), describe a method of encapsulation where pancreatic islets are centrifuged through an alginate solution and a Ba.sup.++ containing medium, which yields alginate-coated islets, the "capsules" having roughly the same size as individual islets and, thereby, effectively eliminating any void volume. There was allegedly no impairment of insulin transport through the alginate coat, although the effect of an additional layer of polylysine was not determined. An interfacial photopolymerization process was reported by Pathak et al., J. Am. Chem. Soc. 114:8311-8312 (1991), for encapsulating pancreatic islets in polyethylene glycol based polymeric microcapsule having a diameter of 500 .mu.m, or for conformal coating as described by Cruise et al., Trans. 19th Ann. Meet. Soc. Biomat. (USA) Abstract 205 (1993). Water insoluble polymers cannot be used as membrane materials by either of these conformal coating/microencapsulation methods.
Encapsulation of mammalian cells in water insoluble synthetic polymer, polyacrylate, for example, by an interfacial precipitation process is described by Sefton and Stevenson, Adv. Polym. Sci. 107:143-197 (1993). Retention of cell viability in vitro and secretion of several bioactive agents have been demonstrated in hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) microcapsules with diameters of approximately 750 .mu.m by Babensee et al., J. Biomed. Mat. Res. 26:1401-1418 (1992), Uludag and Sefton, J. Biomed. Mat. Res. 27:1213-1224 (1993) , and Uludag et al., J. Controll. Rel. 24:3-12 (1993). A limitation of this method is that smaller microcapsules cannot be produced due to the relatively low droplet-shearing force as reported by Crooks et al., J. Biomed. Mat. Res. 24:1241-1262, (1990).
However, the disadvantage exists that prior art methods of making microcapsules less than 500 .mu.m in diameter are not particularly efficient. There is still a need for methods which not only produce small diameter microcapsules, but also produce them efficiently, consistently, and with predictable properties.